We use the Chinese hamster ovary (CHO) cell line (AS52) with a single functional bacterial gpt transgene stably integrated into the genome to study point mutational changes in mammalian cells. Mutants arise at the gpt locus as 6-thioguanine resistant (6TGr) colonies and are characterized as carrying putative point mutations or deletion mutations using the polymerase chain reaction (PCR). Point mutational spectra are then generated using DNA sequence analysis. We have recently implemented methods in the laboratory to simplify the molecular analysis of mutations in mammalian cells. The site of a mutation in a PCR fragment can be quickly identified prior to DNA sequencing using the HOT (hydroxylamine or osmium tetroxide) modification protocol. Such an approach limits the amount of DNA sequencing necessary and allows for the sequence characterization of a substantially greater number of mammalian mutants. We have evaluated the base analog 5-Azacytidine (5AC), the anticancer drug, U73,975 and the human carcinogen, Treosulphan, as well as the suspect active metabolite of treosulphan, diepoxy butane (DEB), as mutagens in AS52 cells. All are potent mutagens and mutational spectra have been generated. Among the point mutations induced by 5AC, there is a substantial bias (>90%) for CG>GC transversions. These data suggest a direct role for 5AC as a direct acting mutagen, most likely through the generation of a 5AC:C mispairing during replication. The drug U73,975 is a less toxic analog of the widely studied antitumor drug, CC-1065. In vitro studies of both drugs have revealed preferred DNA binding sequence motifs. Of 125 U73,975- induced mutants analyzed, 44 (35%) are putative point mutants and 81 (65%) are deletions as analyzed by PCR. Among the point mutants, a hot spot for mutations is observed at a site in the gpt gene containing 3-overlapping consensus drug binding sequences. Treosulphan and DEB induce both deletion mutations (70%) and point mutations (30%). These data suggest that Treosulphan and DEB can induce mutations in mammalian cells via at least two mechanistic pathways. DNA sequence analysis is underway to compare point mutational spectra resulting from treosulphan or DEB treatment. All of these studies are designed to provide insight regarding the mechanisms by which these agents affect genomic integrity.